Note: We use Sigma Product: C2020 as our MALDI matrix, however we also recrystallize before use to purify it further. Sigma also sells a higher-grade MALDI matrix.
| Lab | Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
|---|---|---|---|---|
| Murphy & Metcalf | Formic Acid | Fisher Chemical | A117 | 99.5+%, Optima LC/MS Grade |
| Murphy & Metcalf | α-Cyano-4-hydroxycinnamic acid | Sigma | C2020 | ≥98% (TLC), powder |
| Murphy & Metcalf | Acetonitrile | Fisher | A9551 | LC-MS Ultra CHROMASOLV |
| Murphy & Metcalf | Methanol | Fisher Chemical | A456-4 | LC-MS Grade |
| Murphy & Metcalf | Water | Honeywell | LC365 | LC-MS Grade |
| Murphy & Metcalf | Trifluoroacetic acid | Fisher | A116 | LC-MS Grade |
| Metcalf | Toothpicks | NA | NA | Wood toothpicks are preferred |
| Murphy | Bruker Peptide Calibration standard | Fisher | NC9846988 | Bruker Daltonics 8206195 |
| Murphy | Bruker Daltonics Bacterial test standard | Fisher | NC0884024 | Bruker Daltonics 8604530. Note: This is not IVD BTS, which is for clinic-approved applications. |
| Murphy & Sanchez | 384-spot MALDI plates | Bruker Daltonics | varies | NA |
| Sanchez | Autoflex Speed LRF MALDI-TOF instrument | Bruker Daltonics | NA | Software is being continuously updated to support more vendor instruments |
If you haven’t already, download the Excel template here.
If you don’t have access to Microsoft Excel, you can use any other spreadsheet software such as: Apache OpenOffice™ “Calc”, which can be found at www.openoffice.org. When saving the file, ensure you save it as type “Microsoft Excel 97/2000/XP (.xls or .xlsx)”.
Before applying bacteria:
Sonicate or vortex the solution until no matrix crystals are observed. The matrix is then ready to be applied.
Use matrix solution within 1 week and store unused solid and prepared CHCA matrix between 2-8 °C, in the dark.
When applying bacteria to MALDI plate, your spot should resemble column 2. Column 1 has insufficient cell material, and column 3 has too much cell material
| 1. Apply bacteria directly without any prior chemical treatment. Smear a single bacterial colony in a thin layer directly onto the MALDI target plate using a sterile toothpick. | |
| 2. Leave the Calibration spots empty. | |
| 3. Add 1 µL of 70% formic acid to each spot containing bacteria. Let air dry. | |
| 4. Add 1 µL of MALDI matrix to each bacterial spot, let air dry. |
The MALDI plate should be properly cleaned before shipping. In order to clean the MALDI plate, use the steps below: method adapted from Freiwald & Sauer
Each plate will require multiple calibration spots. For protein data, follow the directions in the pdf link below: - Bruker Biotyper calibration procedure: - https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/InstructionForUse/IFU_Bruker_Bacterial_Test_Standard_Revision_C.pdf
| Insert MALDI plate into the mass spectrometer |
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| Select the appropriate IDBac Method |
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| Under the “AutoXecute” control panel select “New”, which is to the right of “Run” |
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| If it wasn’t automatically detected, select the appropriate MALDI target plate geometry. |
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| Manually determine the minimum laser fluency/power needed and then press “Set initial laser power” before beginning the run |
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| Select “Calibrate with own template” and then select “New” |
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| Follow the directions in the left panel and then select “OK” |
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| Select “Next” |
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| Within the “run parameters” page it is important to ensure the correct methods are selected in the correct places. For small molecule runs change both autoXecute methods to: “IDBac_Small-Molecule_autoX”. For protein runs change both autoXecute methods to: “IDBac_Protein_AutoX.axe”. There are four flexAnalysis methods: The protein or small molecule “Calibrant Calibration” should be selected within the calibration box’s “flexAnalysis Method” pull-down menu. IDBac Protein Calibrant Calibration IDBac Small Molecule Calibrant Calibration. The protein or small molecule “Unknown Sample Calibration” should be selected within the second “flexAnalysis Method” pull-down menu. IDBac Protein Unknown Sample Calibration IDBac Small Molecule Unknown Sample Calibration When you have finished, select “Next” |
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| Select “Save as” and save the sequence run to your data directory. Confirm and select “OK” |
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| Under the “AutoXecute” control panel select “Start automatic Run” |
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Address:
Dr. Brian T. Murphy
900 S. Ashland Ave. M/C 870
MBRB 3114
Chicago, IL 60607
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Contains:
Return Address:
Dr. Brian T. Murphy
900 S. Ashland Ave. M/C 870
MBRB 3114
Chicago, IL 60607